The present invention relates to a novel method to detect epsilon immunoglobulin (IgE). The present invention also includes novel kits to detect IgE as well as methods to produce the detection reagent.
Diagnosis of disease and determination of treatment efficacy are important tools in medicine. In particular, detection of IgE production in an animal can be indicative of disease. Such diseases include, for example, allergy, atopic disease, hyper IgE syndrome, internal parasite infections and B cell neoplasia. In addition, detection of IgE production in an animal following a treatment is indicative of the efficacy of the treatment, such as when using treatments intended to disrupt IgE production.
Until the discovery of the present invention, detection of IgE in samples obtained from non-human animals has been hindered by the absence of suitable reagents for detection of IgE. Various compounds have been used to detect IgE in IgE-containing compositions. In particular, antibodies that bind selectively to epsilon idiotype antibodies (i.e., anti-IgE antibodies) have been used to detect IgE. These anti-IgE antibodies, however, can cross-react with other antibody idiotypes, such as gamma isotype antibodies. The discovery of the present invention includes the use of a Fc epsilon receptor (Fcxcex5R) molecule to detect the presence of IgE in a putative IgE-containing composition. A Fcxcex5R molecule provides an advantage over, for example anti-IgE antibodies, to detect IgE because a Fcxcex5R molecule can bind to an IgE with more specificity (i.e., less idiotype cross-reactivity) and more sensitivity (i.e., affinity) than anti-IgE binding antibodies.
Lowenthal et al., 1993, Annals of Allergy 71:481-484, dog serum can transfer cutaneous reactivity to a human. While it is possible that Lowenthal et al. properly teach the binding of human Fcxcex5R to canine IgE. Lowenthal et al., however, do not provide data defining the particular cellular proteins responsible for the transfer of cutaneous reactivity. As such, a skilled artisan would conclude that the transfer of cutaneous reactivity taught by Lowenthal et al. could be due to a variety of different molecular interactions and that the conclusion drawn by Lowenthal et al. is merely an interpretation. In addition, Lowenthal et al. do not teach the use of purified human Fcxcex5R to detect canine IgE. The subunits of human Fcxcex5R have been known as early as 1988 and have never been used to detect canine, feline or equine IgE. Indeed, U.S. Pat. No. 4,962,035, to Leder et al., issued Oct. 9, 1990, discloses human Fcxcex5R but does not disclose the use of such a human Fcxcex5R to detect human or non-human IgE. The use of purified human Fcxcex5R avoids complications presented by use of Fcxcex5R bound to a cell, such as non-specific binding of the Fcxcex5R-bearing cell due to additional molecules present on the cell membrane. That purified human Fcxcex5R detects non-human IgE is unexpected because inter-species binding between a Fcxcex5R and an IgE is not predictable. For example, human Fcxcex5R binds to rat IgE but rat Fcxcex5R does not bind to human IgE.
The high affinity Fcxcex5R consists of three protein chains, alpha, beta and gamma. Prior investigators have disclosed the nucleic acid sequence for: the alpha chain (Kochan et al., Nucleic Acids Res. 16:3584, 1988; Shimizu et al., Proc. Natl. Acad. Sci. USA 85:1907-1911, 1988; and Pang et al., J. Immunol. 151:6166-6174, 1993); the beta chain (Kuster et al., J. Biol. Chem. 267:12782-12787, 1992); and the gamma chain (Kuster et al., J. Biol. Chem. 265:6448-6452, 1990).
Thus, methods and kits are needed in the art that will provide specific detection of non-human IgE.
The present invention includes detection methods and kits that detect IgE. One embodiment of the present invention is a method to detect IgE comprising: (a) contacting an isolated human Fcxcex5 receptor (Fcxcex5R) molecule with a putative IgE-containing composition under conditions suitable for formation of a Fcxcex5R molecule:IgE complex, wherein the IgE is selected from the group consisting of canine IgE, feline IgE and equine IgE; and (b) determining the presence of IgE by detecting the Fcxcex5R molecule:IgE complex, the presence of the Fcxcex5R molecule:IgE complex indicating the presence of IgE. A preferred Fcxcex5R molecule in which a carbohydrate group of the Fcxcex5R molecule is conjugated to biotin.
Another embodiment of the present invention is a method to detect IgE comprising: (a) contacting a recombinant cell with a putative IgE-containing composition under conditions suitable for formation of a recombinant cell:IgE complex, in which the recombinant cell includes: a recombinant cell expressing a human Fcxcex5R molecule; and a recombinant cell expressing an antibody that binds selectively to an IgE including canine IgE, feline IgE and equine IgE; and (b) determining the presence of IgE by detecting the recombinant cell:IgE complex, the presence of the recombinant cell:IgE complex indicating the presence of IgE. A preferred recombinant cell includes a RBL-hFcxcex5R cell.
Another embodiment of the present invention is a method to detect flea allergy dermatitis comprising: (a) immobilizing a flea allergen on a substrate; (b) contacting the flea allergen with a putative IgE-containing composition under conditions suitable for formation of an antigen:IgE complex bound to said substrate; (c) removing non-bound material from the substrate under conditions that retain antigen:IgE complex binding to the substrate; and (c) detecting the presence of the antigen:IgE complex by contacting the antigen:IgE complex with a Fcxcex5R molecule. Preferably, the flea allergen is a flea saliva antigen and more preferably flea saliva products and/or flea saliva proteins.
The present invention also includes a kit for performing methods of the present invention. One embodiment is a kit for detecting IgE comprising a human Fcxcex5 receptor (Fcxcex5R) molecule and a means for detecting an IgE including canine IgE, feline IgE and equine IgE. Another embodiment is a general allergen kit comprising an allergen common to all regions of the United States and a human Fcxcex5 receptor (Fcxcex5R) molecule. Another embodiment is a kit for detecting flea allergy dermatitis comprising a human Fcxcex5 receptor (Fcxcex5R) molecule and a flea allergen.
Another embodiment of the present invention is an isolated human Fcxcex5 receptor (Fcxcex5R) alpha chain protein, in which a carbohydrate group of the Fcxcex5R alpha chain protein is conjugated to biotin. A preferred Fcxcex5R alpha chain protein comprises PhFcxcex5Rxcex1172-BIOT.